Process of producing dextran



United States Patent 2,801,204 PROCESS OF PRODUCINGDEXTRA'N' Earl R. Kooi, La Grauge, Ill., assiguor to Corn Products Refining Company, New York, N.- Y., a corporation of New Jersey No Drawing. Application April 7, 1954, Serial N0. 421,695

16 Claims. (Cl. 195-31) This invention relates to an improved process for producing dextran. More particularlyv it relates to a process for the enzymatic conversion to dextran ofthe dextrins in partially. hydrolyzed amylaceous materials with enzymes derived from an organism of the genus Acetobacter; e. g., Acetobacter capsulaturm and Acetobacte'r visco'sum.

When Acetobacrer capsulatum or Acetohaazer viscosum is grown ona substrate containingas one of the constituents a carbohydrate, polyhydric alcohol, or asimilar source of organic carbfo'n containing material, three enzyme systems are produced, (1) dextran-dextrinase; which converts dextrins to dextran, (2) an amylase, which hydrolyzes'glucose polymers to glucose, and (3) a glucose oxidase or glucose dehydrogenase which converts glucose to gluconic acid. Thus, when it is desired to prepare dextran by a process involving (1) fermentation of a carbohydrate material, for example, for the accumulation of dextran-dextrinase followed by (2) an enzyme conversion of a partially hydrolyzed amylaceous 'material for the'accumulation of dextran, both of the other enzyme systems are operative during this second period. The result is thatconsiderable quantities of gluconic acid'accumulate during the enzyme conversion period, and unless this acid' is continuouslyneutralized, the pHvalue of the enzyme conversion liquor falls below the optimum value for the accumulation of dextran.

The main object of th'is'invention is to preventthe formation of gluconic acid in such process. This is ac complished by the addition to the enzyme digest of a material which specifically inhibits the glucose oxidase or glucose dehydrogenase, but causes no inhibition of the dextran-dextrinase.

In carrying out the invention an organism such as Acetobacter capsulatum or Acetobacter ViSCOtSllWl is grown for an initial period on a substrate conducive to the formation of dextran-dextrinase. To the culture liquor, adjusted to the pH optimum for dextran-dextrinase activity, there is then added a partially hydrolyzed amylaceous material, such as a starch hydrolyzate, having a D. E. value of about 5 to about 65 and a material, such as sodium cyanide, calcium hypochlorite or sodium bisulfite, which specifically inhibits the formation of gluconic acid by enzymes from the above organism. The mixture is then incubated under conditions conducive to dextran formation. The substrate aforementioned may be a polyhydric alcohol or a partially hydrolyzed amylaceous material or the like, and although the substrate on which the organism is grown is not to be considered a part of this invention, it should be appreciated that the three enzyme systems named above are produced by these two organisms whether the carbon source of the substrate is a partially hydrolyzed amylaceous material or a polyhydric alcohol or other suitable material.

The process of my invention will now be described in further detail with particular reference to partially hydrolyzed starch as both the substrate for the formation of dextran-dextrinase and, also, as the raw material for 2,801,204 Patented July 30,- 1957 "ice the formation of dextran. Starch is hydrolyzed as with acid in conventional manner until the D. E. value (dextrose equivalent, i. e., percent of reducing sugars, on dry basis, calculated as dextrose) is within the range of about 5 to. about 65. The pH-of the resultant starch hydrolyzate 'is adjusted to 4.5 to- 5.5, andthen the hydrolyzate is skimmedor filtered. Thereafter, the solids content is adjusted to about5 percent and thereis added to the concentrated liquor about 0.5: gramper ml. of'an organic nitrogenous material, such as dehydrated yeast extract, corn steep liquor, or the like The pH of the liquor is then adjustedto 5.5 to 6.8 and the liquor is sterilized in knownmanner, e. g., at about C. for 20 minutes. Next the resultant fermentation medium is cooled to about 25 to 3.0 C. and thereafter it is inoculated with a suitable culture grown 10 to 48 hours on the same medium as heretofore described in' amount within the range of about 2 to about 20 percent by volume of' fermentation medium. The medium is agitated'and aerated until dextran-dextrinase activity reaches a substantially maximum'value, for example about 10 to 16 hours. There is then added to the culture'liquor a material which inhibits the enzymaticformation of gluconic acid but does not inhibit the dextran-dextrinase. There may 'alsobe-added an additional quantity'of starch hydrolyzate. The liquor is then incubated-under conditions conducive to-the formation of dextran by the dextran-dextrinase, as for example at pH 4.5 and 30 to 35 C.

Ihave previously discovered that the rate of dextran formation from starch hydrolyzates can be markedly increased by agitation and aeration of the culture liquor. Further that the rate of dextran productionand the con centration at which the substrate can be efficiently con verted to dextran can be efiectively increased by starting the fermentation at a carbohydrate concentration of not more than 10 grams per 100 ml. and adding a concentrated solution of the carbohydrate material after active fermentation has begun. An additional means of insuring rapid dextran formation at high carbohydrate con centrations is to maintain the pH of the culture liquor at about5.5 to 6.8 for the first 10 to'24 hours andthereafter at about 3.5 to 5.0 untiltheifermentation is completed. These discoveries are described in my co-pending application, Serial No. 328,154, filed December 26, 1952.

If an inhibitor for the enzymes producing gluconic acid is not added, the pH of the liquor drops to a value which is not optimum for the action of the dextran-dextrinase. For example, comparativeratesof dextranformation by thedextran-dextrinase enzyme at differentpH'values are as follows:

Rate of Dextran pH Formation in Hours Although the pH during the enzymatic formation of dextran may be maintained at the optimum value by continuous neutralization of the gluconic acid, in either case in the absence of the inhibitor considerable quantities of gluconic acid accumulate. By means of the present invention, it is possible to maintain stable, optimum pH during thecourse of the enzymatic formation of dextran without recourse to additional neutralization. Further, the invention is also advantageous in that the prevention of the formation of gluconic acid results in easy recovery of both dextran and the residual, non-dextran carbohydrate since only two materials need be separated. There is 150 loss of carbohydrate due to formation of gluconic ac1 The invention is particularly applicable to enzymatic conversion of partially hydrolyzed materials, e. g. any starch, to dextran, wherein the enzymes are obtained from cultures of Acetobacter and wherein the enzyme mixture contains, in addition to' the dextran-dextrinase enzyme, an enzyme system which converts glucose to gluconic acid. i The following example will further illustrate the invention but. it should be understood that it in no way limits the invention. Example Acetobacter capsulatum was grown for 17 hours on a medium composed of a 17 D. E. acid hydrolyzate of corn starch together with yeast extract, as above'described. The culture liquor was then adjusted to pH 4.5 and divided into two portions. To each was added sufiicient of a 17 D. E. acid hydrolyzate of corn starch having a pH of 4.5 to bring the total concentration to 13 grams of dry substance per 100 milliliters. To one portion was added about 0.025 gram of sodium bisulfite per 100 milliliters. Both portions-were agitated constantly at 30 C. {Following are the results obtained:

It is readily seen from the above example that sodium bisulfite completely inhibits the formation of gluconic acid and thus results in the maintenance of a constant -pH value throughout the enzymatic formation of dextran.

Sodium cyanide and calcium hypochlorite when used under similar conditions and in the same amount gave comparable results to sodium bisulfite.

I claim:

1. A process for producing dextran which comprises converting the dextrin,'in a partially hydrolyzed amylaceous material, to dextran, by means of dextrandextrinase derived from Acetobacter, in contact with an inhibitor for glucose dehydrogenase from the group consisting of sodium bisulfite, sodium cyanide and callaceous material comprises starch hydrolyzed to a D. E. value within the range of about 5 to about 65.

3. Process according to claim 2 wherein said inhibitor is sodium bisulfite in amount of about 0.025 gram per ml. of substrate.

4. Process according to claim 2 wherein said inhibitor is sodium cyanide in amount of about 0.025 gram per 100 ml. of substrate.

5. Process according to claim 2 wherein said inhibitor is calcium hypochlorite in amount of about 0.025 gram per 100 m1. of substrate.

6. Process according to claim 2 wherein said dextrandextrinase is derived from Acetobacter capsulatum.

7. Process according to claim 2 wherein said dextrandextrinase is derived from Acetobacter viscosum.

8. A process for producing dextran which comprises fermenting a medium comprising a source of nitrogen and a partially hydrolyzed amylaceous material with an organism from Acetobacteriaceae until dextran-dextrinase activity reaches substantially the maximum, thereafter adding an inorganic compound which is an inhibitor for glucose dehydrogenase from the group consisting of sodium bisulfite, sodium cyanide and calcium hypochlorite, and allowing said dextran-dextrinase to convert the substrate to dextran.

, 9. Process according to claim 8 wherein the pH is maintained within the range of 5.5 to 6.8 during the fermenation and within the range of 3.5 to 5.0 during the enzyme conversion.

10. Process of claim 8 wherein said hydrolyzed amylaceous materialcomprises starch hydrolyzed to a D. E. value within the range of about 5 to about 65.

i 11. Process according to claim 8 wherein said inhibitor is sodium bisulfite in amount of about 0.025 gram per 100 ml. of substrate.

12. Process according to claim 8 wherein said inhibitor is sodium cyanide in amount of about 0.025 gram per 100 ml. of substrate.

13. Process according to claim 8 wheerin said inhibitor is calcium hypochlorite in amount of about 0.025 gram per 100 ml. of substrate.

, 14. Process according to claim 8 wherein said dextrandextrinase is derived from Acetobacter capsulatum.

15. Process according to claim 8 wherein said dextrandex-trinase is derived from Acetobacler viscosum.

16. Process according to claim 8 wherein the medium is agitated and aerated during the formation of the dextran-dextrinase.

References Cited in the file of this patent UNITED STATES PATENTS 2,689,816 Kooi Sept. 21, 1954 OTHER REFERENCES Hehre et al.: Proc. Soc. Exp. Biol. Med. 71, 1949, pp. 336.339.

Waksman et al.: Enzymes, 1926, Williams & Wilkins, pp. 53-65. 

1. A PROCESS FOR PRODUCING DEXTRAN WHICH COMPRISES COVERTING THE DEXTRIN, IN A PARTIALLY HYDROLYZED AMYLACEOUS MATERIAL, TO DEXTRIN, BY MEANS OF DEXTRANDEXTRINASE DERIVED FROM ACETOBACTER, IN CONTACT WITH AN INHIBITOR FOR GLUCOSE DEHYDROGENASE FROM THE GROUP CONSISTING OF SODIUM BISULFITE, SODIUM CYANIDE AND CALCIUM HYPOCHLORITE. 